TABLE 1.
Kinetic constants of purine nucleobase uptake in L. mexicana amastigotes and L. major promastigotesa
| Parameter (unit) | Value, mean ± SE (n) for:
|
|||
|---|---|---|---|---|
|
L. mexicana amastigotes
|
L. major promastigotes
|
|||
| [3H]hypoxanthine | [3H]allopurinol | [3H]adenine | [3H]allopurinol | |
| Km (μM) | 1.6 ± 0.4 (4) | 33.6 ± 6.0 (3) | 4.6 ± 0.9 (3) | 54.3 ± 2.9 (3) |
| Vmax (pmol 107 cells−1 s−1) | 0.092 ± 0.057 (4) | 0.051 ± 0.017 (3) | 3.2 ± 0.3 (3) | 0.24 ± 0.06 (3) |
| Ki (μM) with: | ||||
| Adenineb | 4.2 ± 0.8 (5) | |||
| Hypoxanthineb | 3.8 ± 0.6 (3) | 1.3 ± 0.3 (3) | 0.30 ± 0.09 (3) | |
| Guanineb | 1.7 ± 0.1 (4) | 2.8 ± 0.7 (4) | ||
| Xanthineb | 13 ± 2 (3) | 23 ± 8 (3) | ||
| Allopurinolb | 39 ± 6 (3) | 56 ± 1.5 (3) | ||
| Aminopurinolc | 170 ± 20 (4) | NDd | ||
| Purineb | 3.4 ± 0.2 (3) | 6.7 ± 0.4 (3) | ||
| 1-Deazapurinec | 54 ± 5 (3) | 26 ± 4.1 (3) | ||
| 3-Deazaguanine | 130 ± 14 (3) | 48 ± 5.0 (3) | ||
| 6-Thioguanineb | 10 ± 1.2 (3) | 6.2 ± 0.8 (3) | ||
| 7-Deazaguanine | >1,000 (3) | 430 ± 140 (3) | ||
| 9-Deazaguaninee | 204 ± 28 (4) | 204 ± 4 (3) | ||
| Adenosineb | 950 ± 240 (3) | 5,150 ± 550 (3) | ||
| Inosineb | 380 ± 26 (3) | 125 ± 15 (3) | ||
| Guanosineb | 210 ± 48 (3) | 68 ± 17 (4) | ||
Kinetic parameters were determined through competitive inhibition of [3H]hypoxanthine (amastigotes) or [3H]adenine (promastigotes). In a few cases, extrapolation was required due to limitations of solubility of the inhibitor and based on the assumption of a Hill slope of −1 and eventual 100% inhibition. Extrapolation was not attempted when inhibition at the highest inhibitor concentration was <50%. Permeant concentrations were 2 μM (allopurinol) or 0.1 to 0.5 μM (hypoxanthine), except for determinations of Km. Data for L. major promastigotes are from reference 2.
Obtained from Sigma.
Obtained from Aldrich.
ND, not determined.
Gift from Howard Cottam (University of California, San Diego).